Neodymium trinitrate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Scientifically validated test method by ECVAM.

Data source

Reference

Reference Type:

publication

Title:

Assessment of embryotoxicity of compounds in cosmetics by the embryonic stem cell test

Author:

Chen R, Chen J, Cheng S, Qin J, Li W, Zhang L, Jiao H, Yu X, Zhang X, Lahn B T, Xiang A P

Year:

2010

Bibliographic source:

Toxicology Mechanisms and Methods: 20(3): 112-118

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Mouse embryonic stem cell test

Deviations:

not specified

Principles of method if other than guideline:

Neodymium (III) nitrate was assessed for its embryotoxic potential by the embryonic stem cell test (EST). Three end-points were analyzed. The cytotoxicity of the compound on E14 cells and 3T3 cells (IC50 3T3, IC50 ES) were determined by MTT assay, and the inhibition of cardiomyocyte differentiation of embryonic stem cells (ID50) was determined by microscopic observation on day 10.

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Neodymium trinitrate

EC Number:

233-153-9

EC Name:

Neodymium trinitrate

Cas Number:

10045-95-1

Molecular formula:

HNO3.1/3Nd

IUPAC Name:

neodymium trinitrate

Test animals

Species:

other: Murine cell line

Strain:

other: E14 and Balb/c 3T3

Details on test animals or test system and environmental conditions:

Details on cell culture:
Two permanent cell lines were cultured at 37°C and 5% CO2 and routinely passaged three times a week. E14 cells were cultured on mouse embryonic fibroblast feeders (MEF) in a standard ES-cell culture medium containing DMEM supplemented with 10% FCS, 2 mM glutamine, 1% non-essential amino acids, 0.1 mM b-mercaptoethanol, 1000 U/mL leukemia inhibiting factor, 50 U/mL penicillin G, and 50 µg/mL streptomycin. 3T3 fibroblasts were cultured in DMEM containing 10% FCS, 4 mM glutamine, 50 U/mL penicillin G, and 50 µg/mL streptomycin.

Administration / exposure

Route of administration:

other: exposure to cells

Vehicle:

water

Details on exposure:

Cytotoxicity assay:
On day 0, 500 cells in 50 μl routine culture medium without LIF were seeded into each well of a 96-well plate. Culture medium (150 μl) with or without tested compounds were added to each well after 2 h incubation at 37°C and 5% CO2. Each test concentration and positive control was tested in six replicated wells, and seven concentrations were set for each compound in 1:10 dilution. On days 3 and 5, medium was changed with fresh medium containing the respective concentration of tested compounds as on day 0. On day 10, a MTT assay was performed to determine cell viability; 20 μl 5 mg/ml MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) dissolved in PBS was added into each well. After incubation at 37°C and 5% CO2 for 2 h, the medium was removed. Cells were incubated with 130 μl MTT desorbing solution containing 3.49% (v/v) of 20% SDS stock solution and 96.51% (v/v) 2-propanol. Absorbance was measured by a microplate spectrophotometer at 570/630 nm. The cytotoxicity of the tested compounds was determined from the inhibiting concentration of 50% (IC50) obtained through concentration–response curve.

Differentiation assay with ES cells:
750 cells in 20 μl cell culture medium without LIF were seeded as droplet into the lid of a 10-cm Petridish, filled with 10 ml PBS and incubated at 37°C and 5% CO2. After 3 days of hanging drops culture, the cells formed aggregates called embryonic bodies (EBs). The EBs were transferred into a 6-cm bacterial Petri dish and cultured in suspension for 2 days. On day 5, the EBs were placed separately into 24-well plates containing the
appropriated concentration tested compounds, and allowed to attach and outgrow for an additional 5 days. On day 10, the number of wells containing beating myocardial cell areas was counted by using a phase-contrast microscope. Besides solvent control, seven concentrations were set for each compound in 1:10 dilution from 1 mg/ml, except penicillin G from 10 mg/ml and antimony (III) oxide from 10 μg/ml. One 24-well plate containing 24 EBs was used for one test concentration and solvent control. An assay was valid when the solvent control plate had at least 21 wells out of 24 that contained beating myocardial cell areas, and this number were set as 100%. The inhibition of differentiation (ID50) was calculated from the concentration–response curve.

Analytical verification of doses or concentrations:

no

Details on mating procedure:

Not applicable

Duration of treatment / exposure:

10 days

Frequency of treatment:

On days 3 and 5, medium was changed with fresh medium containing the respective concentration of tested compounds as on day 0.

Duration of test:

10 days

No. of animals per sex per dose:

Not applicable

Control animals:

other: not applicable

Details on study design:

seven concentrations were set in 1:10 dilution

Examinations

Maternal examinations:

Not applicable

Ovaries and uterine content:

Not applicable

Fetal examinations:

Not applicable

Statistics:

The statistical analysis was performed using SPSS version 13.0. Data were expressed as mean ± SEM. Each data point represented three independent experiments. Criterion for significance was p<0.05.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Details on maternal toxic effects:
Not examined

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes. Remark: weak

Details on embryotoxic / teratogenic effects:
See field 'Any other information on results incl. tables'.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Positive and negative control results:

5 -fluorouracil and penicillin G were respectively classified as strongly and non-embryotoxic.

Neodymium (III) nitrate hexahydrate:

In the MTT assay, neodymium (III) nitrate hexahydrate showed weak cytotoxicity on 3T3 cells and ES cells (IC50 3T3 541.4 µg/mL, IC50 ES 374.8 µg/mL), and ES cells were more susceptible to the cytotoxic effects of neodymium (III) nitrate hexahydrate than 3T3 cells. The differentiation of ES cells into contracting cardiomyocytes (ID50 188.7 µg/mL) was a little more sensitive than the MTT assay of 3T3 cells. Neodymium (III) nitrate hexahydrate was concluded to possess weak embryotoxicity.

Applicant's summary and conclusion

Conclusions:

Neodymium (III) nitrate hexahydrate was concluded to possess weak embryotoxicity in an in vitro test. However this study is not adequate to support hazard classification. In addition, no NOAEL or other dose-response information required for risk assessment purposes is provided by a such a test